Blood-coagulation-promoting products and methods of preparing them

ABSTRACT

Blood-coagulation-promoting products substantially free of thrombin are prepared from human blood plasma by contacting a human blood plasma fraction containing coagulation Factors II, VII, IX and X with an anion exchanger to adsorb the coagulation Factors, which are subsequently eluted from the anion exchanger. The eluate is treated to generate a substance having Factor VIII Inhibitor Bypassing Activity and being substantially free of thrombin, and activated Factor X.

BACKGROUND OF THE INVENTION

1. Field of the Invention:

This invention relates to and has among its objects novelblood-coagulation promoting products and methods of preparing them. Itis a particular object of the invention to provideblood-coagulation-promoting products that contain substantial amounts ofFactor VIII Inhibitor Bypassing Activity substance but are substantiallyfree of thrombin. Further objects of the invention will be evident fromthe following description.

2. Description of the Prior Art:

There are estimated to be 100,000 cases of congential hemophilia in theUnited States. The two predominant types of hemophilia are Hemophilia Aand Hemophilia B. A cure for this congenital disease is not available;however, treatment of the disease has been possible by supplyinghemophiliacs with active blood coagulation factors by intravenousadministration. An early approach was to administer fresh blood or freshplasma to temporarily correct the defect of the deficient subject. Morerecently, concentrates containing particular coagulation factors lackingin different hemophiliacs have been isolated from blood plasma. Sincethese preparations contain the blood coagulation factors in concentratedform, much lower amounts may be used to achieve rapid stoppage ofbleeding, and therapy is more specific than with plasma.

One such preparation is called antihemophilic factor (AHF) or FactorVIII for treatment of Hemophilia A. Factor IX (Antihemophilic Factor B)preparations, such as those described in U.S. Pat. No. 3,717,708, areused for treatment of Hemophilia B patients.

Approximately 15 to 25% of Hemophilia A patients have an inhibitor toFactor VIII. Consequently, when Factor VIII preparations are given tothese patients, the effect of Factor VIII is negated because ofinhibition of its activity. This inhibitor may be removed by plasmaexchange of the patient's plasma for that of a healthy donor or forplasma substitutes. However, the complexity of the exchange proceduremakes this approach very undesirable. Animal source Factor VIII can beused, but it shortly elicits a foreign protein response which negatesits usefulness.

Recently, in U.S. Pat. No. 4,160,025, (hereinafter '025) there wasdescribed a new blood-coagulation-promoting preparation from human bloodplasma, which contains a blood coagulation effective substance causing abypassing of the Factor VIII inhibitor. This new substance was termedFEIBA for "Factor VIII Inhibitor Bypassing Activity" substance. It wasnoted in '025 that certain "activated" prothrombin complex concentrates,containing Factor IX among others, had been used successfully incontrolling bleeding of Factor VIII inhibitor patients. The activationof these prothrombin concentrates was probably due to unknownimpurities. The FEIBA of '025 was a preparation which safeguarded in arepeatable and deliberate manner a generation of Factor VIII inhibitorbypassing activity. FEIBA was clinically effective and compatiblewithout undesired side effects.

To prepare FEIBA by the method of '025 human plasma containing citrateions is treated in the absence of free calcium ions with water-insolubleinorganic coagulation-physiologically-surface-active substances, such assilica gel or kaolin, to generate a FEIBA substance. Water insolublesubstances are separated from the FEIBA-containing material, which isthen treated with a basic ion exchanger, such as adiethylaminoethyl-group-containing high molecular weight substance, toadsorb the FEIBA substance thereon together with coagulation Factors II,VII, IX, and X. The adsorbed material is eluted and concentrated. Thenew substance with FEIB-activity is a protein with a higher molecularweight (100,000) than that of unactivated Factors II, VII, IX, and X(70,000) and contains some thrombin.

It is known that calcium ions in conjunction with other materials suchas phospholipids and activated Factor X assist in the activation ofBovine Factor VII (Radcliffe et al., The Journal of BiologicalChemistry, 1975, Vol. 250, No. 2, pp. 388-395, and Radcliffe et al.,ibid., 1976, Vol. 251, No. 16, pp. 4797-4802). Further, it is well-knownthat the action of calcium ions on a mixture of the above coagulationFactors produces substantial amounts of thrombin, which is anundesirable substance when present in materials to be infused intohumans. Thus, those skilled in the art have not considered and would notconsider the possibility of generating a FEIBA substance by usingcalcium ions either alone or in conjunction with other agents (e.g.'025).

Currently, there is a Factor VII Inhibitor Bypassing Activity substanceof anti-inhibitor coagulant complex marketed by Hyland Laboratories(Costa Mesa, Calif.) under the name AUTOPLEX®. The FEIBA:thrombin ratioof this material is about 10:1, FEIBA:Factor II is about 10:1,FEIBA:Factor VII is about 2:9, FEIBA:Factor IX is about 1:2, andFEIBA:Factor X is about 5:1. (The above sample was analyzed according tothe assays outlined hereinbelow in the Examples section).

SUMMARY OF THE INVENTION

We have discovered methods for preparing novel Factor VIII InhibitorBypassing Activity (hereinafter referred to as FEIBA) substances withFactor VIII Inhibitor Bypassing Activity (hereinafter referred to asFEIB-activity). The substances of the invention also may be termedAntihemophilic Factor Inhibitor Bypassing Complexes havingAntihemophilic Factor Inhibitor Bypassing Activity or anti-inhibitorcoagulant complex.

The novel FEIBA substances include coagulation Factors II, VII, IX, andX and are substantially free of thrombin and activated Factor X.Generally, in preparing the FEIBA substances of the invention a humanblood plasma fraction containing coagulation Factors II, VII, IX, and Xis contacted with an anion (basic ion) exchanger on which thecoagulation Factors are adsorbed. The specific substance or substancesadsorbed and subsequently activated to FEIBA are not known. It may bethat the mixture of coagulation Factors II, VII, IX, and X contains aspecific FEIBA substance precursor which is adsorbed on the ionexchanger together with the coagulation Factors and then activated to aFEIBA substance. Or, the coagulation Factors themselves may constitutesuch a FEIBA precursor. In any event, applicants have demonstrated thatFEIBA substances can be prepared as long as one begins with a mixture ofthe aforenamed coagulation Factors.

The ion exchanger is selectively washed to remove inhibitors to thegeneration of FEIBA substance without removing the coagulation Factorswhich are subsequently eluted from the basic ion exchanger. The eluateis treated in a number of alternative ways to generate a FEIBAsubstance. In accordance with one aspect of the invention the adsorbedcoagulation Factors are eluted from the basic ion exchanger using anaqueous solution with a certain ionic strength. Surprisingly, we havediscovered that calcium ions can be employed to generate substantialamounts of FEIBA without generation of substantial amounts of thrombin.The eluate is mixed with a source of calcium ions in an amountsufficient to generate substantial amounts of a FEIBA substance butinsufficient to generate substantial amounts of thrombin and held at atemperature and pH and for a time sufficient to generate a FEIBAsubstance.

Alternatively, in one embodiment of the invention the eluateunexpectedly can be held for a period of time and at a temperature andpH sufficient to generate substantial amounts of a FEIBA substance inthe absence of calcium ions or other activating material. The FEIBAsubstance produced in this manner is characterized particularly as beingfree of thrombin.

In accordance with another aspect of the present invention the adsorbedcoagulation Factors are eluted from the basic ion exchanger usingaqueous ammonium bicarbonate. The eluate, after treatment to removeammonium bicarbonate, is mixed with a source of calcium ions in anamount sufficient to generate FEIBA and held at a temperature and pH andfor a time sufficient to generate a FEIBA substance.

A primary advantage of the invention is that FEIBA substances can beproduced in substantial amounts and they are substantially free ofthrombin. The injection of thrombin into a human is considered highlydangerous. Although it is possible to neutralize thrombin activity withheparin in the presence of antithrombin III, heparin is potentiallyhazardous to the patient and interferes with the assay of theaforementioned coagulation Factors, which in turn complicates monitoringof infusion into a patient.

Another advantage of the invention is that the so-produced FEIBAsubstance is substantially free of the activated form of Factor X. Thus,the danger of precipitating intravascular coagulation is reduced.

A particular advantage of the invention is that a FEIBA substance isgenerated in substantial amounts in the last step of the invention.Thus, one has a choice as to which product is to be manufactured, e.g.,a Factor IX concentrate or a FEIBA substance, until the end of theprocess. In the method of '025 a FEIBA substance is generated in thefirst step of the patented process, which precludes a choice in thepreparation of other useful coagulation products.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The product of the invention is prepared from human blood plasma. Thepreponderance of blood generally taken and available for transfusion isprotected from coagulation by treatment with a citrate anticoagulantwhich allows the blood to be utilized for only a limited period of time.After this limited time expires, this blood must be discarded or it canbe made available for fractionation into certain useful components. As amatter of fact, the major source of blood converted to plasma forfractionation comes from blood collected by plasmapheresis which hasbeen protected by citrate anticoagulation. It is, therefore, importantthat processes for the fractionation of plasma be directed to citratepreserved blood, to which the instant method is.

As mentioned above, in the first step in the process of the invention ahuman blood plasma fraction containing coagulation Factors II, VII, IX,and X is contacted with a basic ion exchanger on which the coagulationFactors are adsorbed. A number of fractionation methods have beenapplied to human blood plasma, and these methods are summarized in "ThePlasma Proteins", second edition, Vol. III, pages 548-550, AcademicPress, New York, N.Y. (1977). The preferred starting material in theprocess of the invention is Effluent I from a Cohn fractionation method(U.S. Pat. No. 2,390,074 and J. Am. Chem. Soc., Vol. 68, page 459(1946). Effluent I is contacted with an anionic or basic ion exchanger,for example, DEAE (diethylaminoethyl) Sephadex® consisting ofcross-linked dextran chains with diethylaminoethyl groups attached byether linkages to the glucose units of the polysaccharide chains,supplied by Pharmacia Fine Chemicals, Inc., Piscataway, New Market,N.J., to adsorb the aforementioned coagulation Factors. The spentEffluent I is returned to the plasma fractionation process so that noneof the other plasma components is wasted.

Generally, contact between Effluent I and DEAE Sephadex is achieved byforming a bed of freshly equilibrated DEAE Sephadex and passing EffluentI therethrough. Approximately 10 g. (wet weight) of DEAE Sephadex perliter of Effluent I is used although the amounts employed may be variedover a wide range.

The ion exchanger is washed next to remove inhibitors to the generationof a FEIBA substance. Thus, the DEAE Sephadex bed can be treated withabout 0.1-0.3 M ammonium bicarbonate at pH 7.0-7.8 until theconcentration of protein in the eluate is less than about 4 mg/ml. Theconcentration of the wash solution, in general, should be sufficient toremove inhibitors to the generation of a FEIBA substance butinsufficient to remove the adsorbed coagulation Factors. It is withinthe compass of the invention to wash the DEAE Sephadex more than once.

The above-described steps are similar to those set forth in U.S. Pat.No. 3,717,708, which is incorporated herein by reference.

In accordance with the instant invention the washed DEAE Sephadex withthe aforementioned coagulation Factors adsorbed thereon can be treatedin one of two ways to elute the adsorbed substances. In one aspect ofthe invention (Method A) the DEAE Sephadex can be eluted with ammoniumbicarbonate having a concentration of about 0.5-2.0 M preferably about0.75 M. Generally, the eluting solution should have a concentration ofreadily removable salt, such as ammonium bicarbonate, sufficient toelute the adsorbed coagulation Factors but insufficient to remove otheradsorbed proteins. The pH of the eluting solution should be about 7-9.

The eluate is treated next to remove the salt, e.g., ammoniumbicarbonate, from the solution. To this end the eluate may be dialyzedor diafiltered. Alternatively, the eluate may be lyophilized duringwhich procedure the ammonium bicarbonate is volatilized and removed fromthe product.

The eluate, or lyophilized product, can be constituted, orreconstituted, in a solution containing about 0.05-0.20 M sodiumchloride and about 0.05-0.15 M sodium citrate, preferably physiologicalconcentrations. The solution may contain a buffering agent such asTRIS-chloride or the like.

To this solution of coagulation Factors is added a source of calciumions in an amount sufficient to generate a FEIBA substance butinsufficient to generate substantial amounts of thrombin. Usually,enough calcium ions are added such that the effective calcium ionconcentration, i.e., the level of calcium ions not bound tonon-proteinous material, is about 0.0005-0.0008 mole per liter (M) ofeluate.

As the source of calcium ions one may use any calcium containingmaterial that will produce free calcium ions in an aqueous medium and bephysiologically compatible with the final product. Thus, the calciumsource may be, by way of example and not limitation, calcium chloride,calcium acetate, calcium glycinate, and so forth.

The solution of coagulation Factors with added calcium ions is held at atemperature and pH and for a period of time sufficient to generate aFEIBA substance. The pH of the solution should be about 6 to 9,preferably about 7 to 8, and optimally at about 7.6. The pH may beadjusted by means of the aforementioned buffering agent or by adding anappropriate amount of a physiologically compatible acid, such ashydrochloric acid or the like, to achieve the desired pH.

The temperature at which the solution is held is usually within therange of about 0° to 30° C., preferably about 5°-20° C., and morepreferably about 8°-14° C. Maximum generation of FEIBA substance occursat a temperature of about 10° C.; surprisingly, in most cases at leasttwice as much FEIB-activity is generated at about 10° C. than at about5° C. or about 20° C.

The solution is held for a period of about 4 to 48 hours, preferablyabout 12 to 36 hours to allow a sufficient level of FEIBA substance tobe generated. In a typical example, eluate of pH 7.5, treated with0.0007 molar calcium chloride at 10° C. for 24 hours yielded 170 Unitsper milliliter (U/ml) of FEIB-activity as defined and determined by amodified '025 assay described hereinbelow.

It is generally desirable to treat the eluate after generation of FEIBAsubstance with an agent which scavenges calcium ions and thereby stopsthe generation of FEIBA and thrombin. For this purpose one may use achelating agent that removes positive ions such as Chelex®-100 (BioRadLaboratories, Inc., Richmond, Calif.), Amberlite (Rohm and Haas Co.,Philadelphia, Penn.), sodium citrate, sodium phosphate, or the like inan amount sufficient to remove the calcium ion and prevent furthergeneration of a FEIBA substance. The calcium scavenging agent ischaracterized by its ability to remove calcium ions and its ease ofremoval from the solution. Thus, the preferred agent is an ionexchanger, such as Chelex®-100 in a concentration of about 3-4 g per 100ml of a 5% protein solution, or a biologically compatible chelatingagent.

Following removal of calcium ions the solution may be treated to removewater therefrom by procedures well known in the art. For instance, themixture can be freeze-dried or ultrafiltered and then freeze-dried.Furthermore, the mixture can be sterile filtered by conventional methodsprior to water removal.

The dried compositions containing FEIBA substance can be formulated intopharmaceutical preparations for therapeutic, diagnostic, or other uses.To prepare them for intravenous administration the compositions aredissolved usually in water containing physiologically compatiblesubstances such as sodium chloride, glycine, and the like and having abuffered pH compatible with physiological conditions. Generally,guidelines for intravenously administered compositions are establishedby governmental regulations.

The product prepared in accordance with Method A is characterized assubstantially free of thrombin, and activated Factor X, comprisingcoagulation Factors II, VII, IX, and X and having a Factor VIIIInhibitor Bypassing Activity of at least about 60 U/ml.

The term "substantially free of thrombin" means that the Method Aproduct contains less than 1.5 U/ml of thrombin activity and generallyhas a FEIBA:thrombin ratio of at least about 50:1, e.g., 50:1 to 800:1or more.

The FEIBA substance of Method A is characterized in that theFEIBA:Factor Xa ratio is at least about 45:1, e.g., about 45:1 to 200:1or more (Factor Xa=activated Factor X).

The coagulation Factors are present in the product of Method A generallyin the following ratio--FEIBA:Coagulation Factors, about 10:1 to 0.1:1,preferably as follows--FEIBA:Factor II, about 1.5:1 to 6:1; FEIBA:FactorVII, about 0.5:1 to 1.5:1; FEIBA:Factor IX, about 1:1 to 2:1; andFEIBA:Factor X, about 1.5:1 to 7:1.

As mentioned above, it is within the purview of the invention tolyophilize the eluate from the DEAE Sephadex® to yield an ammoniumbicarbonate-free protein powder as disclosed in U.S. Pat. No. 3,717,708.Thus, another starting material in this method of the invention is theproduct of U.S. Pat. No. 3,717,708 either in dry form or reconstitutedin a buffer solution such as described in U.S. Pat. No. 3,717,708 orhereinabove. If the dry product of U.S. Pat. No. 3,717,708 is employed,it is reconstituted in a buffer solution such as, for example, aqueoussodium citrate-sodium chloride, Tris (hydroxymethyl) aminomethanehydrochloride (TRIS-chloride) and the like. A source of calcium ions isadded to achieve the aforementioned critical concentration. It isimportant to note that some materials, e.g., citrate ions, formcomplexes with calcium ions thereby reducing the concentration of freecalcium ions. Thus, one must add calcium ions until the concentration offree or uncomplexed calcium ions is about 0.0005 to 0.0008 mole perliter. The concentration of free calcium ions can be determined byprocedures standard in the art. The temperature, pH, and holding periodare those described above.

In another aspect of the invention (Method B) the washed DEAE Sephadex®with coagulation Factors II, VII, IX, and X adsorbed thereon can bewashed further with an aqueous solution of an inorganic salt having anionic strength (I) sufficient to remove inhibitors to the generation ofa FEIBA substance but insufficient to elute the adsorbed coagulationFactors. Usually, the I of the aqueous solution should be less thanabout 0.3, preferably about 0.2. Typical examples of aqueous solutionsthat may be used are 0.1-0.3 M aqueous sodium chloride (I=0.1-0.3),TRIS-chloride (I=0.1-0.3), and the like.

Following this washing step the basic ion exchanger is treated to elutethe adsorbed coagulation Factors. To this end the basic ion exchanger iscontacted with an aqueous solution having an ionic strength sufficientto remove the adsorbed coagulation Factors but insufficient to removeother adsorbed proteins. The I of aqueous solutions employed for thispurpose should be, generally, greater than about 0.35, preferably 0.55,and within the range of about 0.35-2.0. Aqueous solutions falling withinthe above description are, by way of example and not limitation,0.35-2.0 M aqueous sodium chloride (I=0.35-2.0), and so forth. Ingeneral, the aforementioned wash and elution solutions should contain asalt that is physiologically compatible with the final product.

The eluate containing the aforesaid coagulation Factors is treated toadjust the concentration of salt therein to 0.2 or less. Furthermore, ina preferred embodiment of the invention the Factors II, VII, IX, Xconcentration (FC) should be at least about 10 U/ml, preferably, atleast about 50 U/ml. The aforementioned objectives for I and FC may beaccomplished for example, using a combination of ultrafiltration anddiafiltration as is known in the art. Other modes of operation toachieve the above I and FC will be suggested to the skilled artisan. Itis important to note that an FC of about 10 is necessary to generatesubstantial amounts, at least about 60 U/ml, of FEIBA substance,although an FC less than 10 would yield a product comparable to knownproducts.

Next, the eluate is treated to generate a FEIBA substance. Aparticularly unexpected feature of Method B is that FEIB-activity can begenerated in the absence of any added agents. Thus, the eluate simply isheld at a pH and temperature and for a period of time sufficient togenerate a FEIBA substance. In general, the above parameters of pH,temperature, and time are the same as those recited hereinabove inMethod A. Typically, eluate held at a temperature of 10° C. and a pH of7.5-7.9 for a period of 24 hours contained about 80 U/ml FEIB-activity.The FEIBA product produced in accordance with this procedure ischaracterized as being essentially free of thrombin, i.e., containingless than 0.5 Units per ml of thrombin. Generally the FEIBA:thrombinratio in products made in accordance with this aspect of Method B isabout 1000:1 or higher. Usually, the presence of thrombin in suchsamples cannot be detected. The ratios of FEIBA to coagulation FactorsII, VII, IX, and X and to Factor Xa are similar to those for the MethodA FEIBA substance.

It is important to note that the above-mentioned mode of activationwithout the use of extraneous agents is peculiar to Method B of theinvention. If the procedure of Method A is followed, a FEIBA substancewill not be generated in the absence of added calcium ions. It isbelieved that the Method B activation in the absence of added agentsresults from the presence of endogenous calcium ions present in thesource plasma. However, activation may be stimulated by other agents andit is not meant to limit the invention by any particular explanation. Wehave demonstrated that a FEIBA substance can be obtained repeatably andreliably in significant, i.e., therapeutically beneficial, amounts inthe absence of added activators by Method B of the invention.

The eluate of Method B also can be mixed with an activating agent suchas a source of calcium ions to generate a FEIBA substance. However, onesurprising feature of this aspect of the invention is that the amount ofcalcium ions needed to generate FEIB-activity at levels equivalent tothat generated in Method A is about ten-fold less in Method B. Indeed,excellent levels, at least 100-200 U/ml or more, of FEIB-activity can beobtained at concentrations of free calcium ions of about 0.000025-0.0005mole per liter preferably 0.000025-0.00005 mole per liter. Theparameters of temperature, pH, and time are the same as those describedabove. In a typical example, eluate mixed with 0.00005 mole per litercalcium ions at pH 7.5-7.9, 10° C., for 24 hours yielded about 240 U/mlFEIB-activity.

The ratio of FEIBA to thrombin, to Factor Xa, and to the coagulationFactors II, VII, IX, and X are approximately the same for this productas for the FEIBA substance of Method A.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples.

ASSAY METHODS

FEIBA Substance: The procedure used was essentially that of '025 and ofPepper et al., Brit, J. Haem., Vol. 36, pages 573-583 (1977) designed toassay in vitro FEIB-activity, which is defined as a shortening of theactivated partial thromboplastin time (APTT) of a Factor VIII inhibitorplasma. The assay was carried out by manual tube-tilting technique.

The reagents used were:

(a) Factor VIII inhibitor plasma obtained from George King Bio-Medical,Overland Park, Kans.

(b) The buffer for dilution was a mixture containing 0.06 MTRIS-chloride, 0.09 M sodium chloride, and 0.5% Human Serum Albumin(Cutter Laboratories, Inc., Berkeley, Calif.) at pH 7.40±0.05.

(c) Kaolin suspension (3 mg/ml) was prepared in physiological saline.The suspension must be shaken vigorously prior to pipetting.

(d) Rabbit Brain Cephalin, Sigma Chemical Co., St. Louis, Mo. wasreconstituted with physiological saline according to directions and waskept frozen in a small volume in a plastic tube. Before carrying out theassay procedure, frozen cephalin was thawed, diluted 30-fold withsaline, maintained at 37° C. for 10 minutes, and then kept at roomtemperature, whereat it is stable for several hours.

In the assay 0.1 ml Factor VIII inhibitor plasma, 0.1 ml buffer, and 0.1ml kaolin suspension were added to a 10×75 mm. glass tube in succession.The reagents were mixed and placed in a 37° C. bath with simultaneousstarting of a stopwatch. Fifteen seconds prior to the completion of theincubation, cephalin was added, and after 10 minutes 0.1 ml of 0.025 Mcalcium chloride was added. The contents were mixed thoroughly, and thetube was held undisturbed. After 30 seconds the tube was tilted at10-second intervals; the content of the tube became more viscous thusrequiring more frequent tilting. A sample that reduced blank time tohalf is defined (in '025) to contain one unit of FEIB-activity.

Factors II and VII: Factor II and Factor VII were assayed by the methodof Owren described in the Scand. J. Clin. and Lab. Investigation, Vol.1, page 81 (1949).

Factors X and Xa: Factor X and Factor Xa were assayed by the method ofBachman et al., described in Thromb. Diath. Haemorrh., Vol. 2, page 24(1958).

Thrombin: The assay procedure employed was described by Fenton II etal., in Thrombosis Res., Vol. 4, pages 809-817 (1974).

Factors IX and VIII: Modification of the procedures described byLangdell et al. (partial thromboplastin time technique), J. Lab. Clin.Med., Vol. 41, pages 637-647 (1953) and by Proctor et al. (kaolinclotting time method) Amer. J. Clin. Path., Vol. 36, page 212 (1961)were employed. Platelet Factor 3 was supplied by a cephalin suspension.Maximum surface contact activation was achieved with Celite® powder. Allother clotting factors (except Factor IX or Factor VIII) were suppliedby a substrate comprising plasma from a patient severely deficient inFactor IX or Factor VIII mixed with barium sulfate adsorbed beef plasma.Quantitation of an unknown specimen was made by comparing its clottingtime in the test with that achieved by dilutions of a normal standard.

The exact assay procedure is the same for both Factor IX and Factor VIIIexcept that the activator in Factor IX assay is Platelin® Plus Activatorinstead of Automated APTT reagent (General Diagnostics, Inc., MorrisPlains, N.J.).

EXAMPLE 1

The lyophilized product of U.S. Pat. No. 3,717,708 was dissolved in abuffer mixture containing 0.05 M TRIS and 0.10 M sodium chloride (pH7.6) to a level of 5% (5 g. of protein per 100 ml of solution). Thesolution was centrifuged at 10,000×g and cooled to 10° C. Calciumchloride was added to the solution to a concentration of 0.0007 M.Aliquots of the solution were assayed by the above method at selectedtime intervals up to 24 hours.

To the solution (100 ml) was added 4 g. of Chelex®-100. The solution wasstirred for about 20 min. and the Chelex®-100 was removed by filtration.The solution containing the generated FEIBA substance was assayedaccording to the above-described procedures. The results are outlined inTable 1A.

After being assayed the solution was sterile-filtered and lyophilized togive a dry product (6 g).

                  TABLE 1A                                                        ______________________________________                                        Time          FEIBA    Thrombin                                               (hrs)         (U/ml)   (U/ml)                                                 ______________________________________                                        0             6        0                                                      4             11       0.10                                                   8             16       0.16                                                   12            49       0.27                                                   16            85       0.48                                                   20            120      0.73                                                   24            170      1.1                                                    ______________________________________                                    

The above-described procedure was repeated with the exception that theproduct was assayed only after 24 hours had passed. The results arefound in Table 1B.

                  TABLE 1B                                                        ______________________________________                                        FEIBA     Factors (U/ml)     Thrombin                                         (U/ml)    II    VII      IX  X     Xa  (U/ml)                                 ______________________________________                                        118       33    93       98  46    2   0.6                                    ______________________________________                                    

EXAMPLE 2

The procedure of Example 1 was followed with the following exceptions:(1) the pH of the solutions was varied over the range of 7.1 to 7.7 and(2) each run was made for a period of 24 hours. The results aresummarized in Table 2A.

                  TABLE 2A                                                        ______________________________________                                                   FEIB-Activity                                                             pH  (sec.).sup.a                                                       ______________________________________                                               7.1 120                                                                       7.2 111                                                                       7.3 113                                                                       7.4 108                                                                       7.5 107                                                                ______________________________________                                         .sup.a Clotting time at dilution 1:10; a smaller value for clotting time      indicates greater FEIBactivity.                                          

The above experiment was repeated and the results are summarized inTable 2B.

                  TABLE 2B                                                        ______________________________________                                                   FEIB-Activity                                                             pH  (sec.)                                                             ______________________________________                                               7.5 111                                                                       7.6 108                                                                       7.7 110                                                                ______________________________________                                    

EXAMPLE 3

The procedure of Example 1 was followed with the exceptions noted below:(1) the temperature at which the material was held was varied (5° C.,10° C., and 22° C., respectively) and (2) the holding time at eachtemperature was 24 hours. The results are outlined in Table 3.

                  TABLE 3                                                         ______________________________________                                                          FEIB-Activity                                               Temperature       (sec.).sup.a                                                (°C.)      1:10   1:20                                                 ______________________________________                                         5                110    --                                                   10                101    110.sup.b                                            22                110    --                                                   ______________________________________                                         .sup.a Clotting time at dilution 1:10 and 1:20                                .sup.b Clotting time of 110 sec. at dilution 1:20 for sample run at           10° C. is same as clotting time at dilution 1:10 for sample run at     5 and 22° C.; this indicates that the sample run at 10° C.      was twice as active as those run at 5 and 22° C.                  

EXAMPLE 4

Konyne® Factor IX Complex (Human) (Cutter Laboratories, Inc., Berkeley,Calif.) was reconstituted according to directions. The sample wasbuffered with 0.05 M sodium citrate and 0.088 M sodium chloride; proteincontent was 2.5%. The solution was brought to 10° C. and calciumchloride was added to a concentration of 0.012 M (free calcium ionconcentration 0.0007 M). Aliquots of the solution were assayed asdescribed above at time intervals of 0, 4, 8, 12, 16, 20, and 24 hours;the results are found in Table 4.

Chelex®-100 (1 g) was added to the solution, which was stirred for 20minutes and then was filtered. Following removal of the Chelex®-100 thesolution was sterile-filtered and lyophilized to give a dry product.

                  TABLE 4                                                         ______________________________________                                        Time          FEIBA    Thrombin                                               (hrs)         (U/ml)   (U/ml)                                                 ______________________________________                                        0              0.sup.a 0                                                      4             10.sup.a 0.11                                                   8             11.sup.a 0.11                                                   12            17.sup.a 0.15                                                   16            40       0.19                                                   20            56       0.25                                                   24            66       0.31                                                   ______________________________________                                         .sup.a See Table 11, footnote 6.                                         

EXAMPLE 5

The procedure of Example 1 was used with the exception that: (1) thecalcium ion concentration was varied and (2) the solution was held for24 hours after addition of calcium before the foregoing assays wereperformed. Table 5 contains the results in outline form.

                  TABLE 5                                                         ______________________________________                                        Calcium ion                                                                   Concentration   FEIBA                                                         (mole/liter)    (U/ml)                                                        ______________________________________                                        0                44                                                           0.00052          50                                                           0.00068         100                                                           0.00075         221                                                           0.00083.sup.a   Clot                                                          ______________________________________                                         .sup.a Not in accordance with the invention but provided for purposes of      comparison.                                                              

EXAMPLE 6

A. The lyophilized U.S. Pat. No. 3,717,708 product was dissolved in abuffer mixture containing 0.05 M TRIS and 0.10 M sodium chloride (pH7.6) to a level of 5% on a weight to volume basis. After the solutionwas centrifuged at 10,000×g and cooled to 10° C., kaolin was added togive a 1% kaolin suspension. The mixture was held at 10° C. for 24 hoursand assayed according to the aforementioned procedures.

B. The procedure in A above was repeated with the exception that Celite®(Johns-Manville Co., New York, N.Y.) was added to a 1% level in place ofkaolin.

C. The procedure in A above was repeated with the exception that thesolution was made 0.0007 M in calcium ions by addition of calciumchloride in place of kaolin.

                  TABLE 6                                                         ______________________________________                                                                  FEIBA                                               Run         Generating Material                                                                         (U/ml)                                              ______________________________________                                        C           0.0007 M Ca Cl.sub.2                                                                        200                                                 B.sup.a     1% koalin     50                                                  A.sup.a     1% Celite®                                                                              76                                                  Control.sup.a                                                                             None          35                                                  ______________________________________                                         .sup.a Not in accordance with the invention but provided for purposes of      comparison. Runs B and A are in accordance with the method of '025.      

EXAMPLE 7

Effluent I (30 l.) was contacted with 300 g. of DEAE Sephadex® gel andmixed together. After 2 hours the mixture was filtered to give 300 g. ofgel, which was washed sequentially with 3 l. of 0.2 M ammoniumbicarbonate, 2 l. of 0.3 M ammonium bicarbonate, and 3 l. of 0.2 Msodium chloride. (Effluent I=Cohn Supernatant I).

After washing, 2 l. of 0.55 M sodium chloride (I=0.55) was applied tothe gel to give an eluate (A₂₈₀ =12.80). The eluate was concentrated toA₂₈₀ to about 50 by ultrafiltration and diafiltered against 0.05 M TRISand 0.1 M sodium chloride (pH 7.6) buffer to a protein concentration ofA₂₈₀ =69.5.

Calcium chloride was added to the eluate (cooled to 10° C.) to aconcentration of 0.0005 M, and the eluate was held at 10° C. for 24hours. Then, Chelex®-100 resin was mixed with the eluate to a level of10% on a weight to volume (w/v) basis. The eluate was filtered to removethe resin and sodium citrate was added thereto to a level of 0.01 M.

The eluate was analyzed as described above to give the results outlinedin Table 7.

                  TABLE 7                                                         ______________________________________                                                           Factors                                                              FEIBA    Thrombin  IX     VII                                       Sample    (U/ml)   (U/ml)    (U/ml) (U/ml)                                                                              A.sub.280                           ______________________________________                                        Post-activation                                                                         185      2.9.sup.a N.A..sup.b                                                                           N.A..sup.b                                                                          69.5                                Pre-activation                                                                          17.8     None      71.5   46.5  69.5                                                   detected                                                   ______________________________________                                         .sup.a 1:100 dilution                                                         .sup.b N.A. = not assayed                                                

EXAMPLE 8

The procedure employed was the same as that of Example 7 with adifferent batch of Effluent I. The results are summarized in Table 8.

                  TABLE 8                                                         ______________________________________                                                           Factors                                                              FEIBA    Thrombin  IX     VII                                       Sample    (U/ml)   (U/ml)    (U/ml) (U/ml)                                                                              A.sub.280                           ______________________________________                                        Post-activation                                                                         120      1.5.sup.a N.A..sup.b                                                                           N.A..sup.b                                                                          31.3                                Pre-activation                                                                           10      None      37.3   19.0                                                         detected                                                   ______________________________________                                         .sup.a 1:50 dilution                                                          .sup.b N.A. = not assayed                                                

EXAMPLE 9

The procedure of Example 7 was repeated using 1 kg of DEAE Sephadex. Theprotein concentration (A₂₈₀) during activation was 41.5. Table 9Acontains a summary of the results.

                                      TABLE 9A                                    __________________________________________________________________________               Factors                                                                  FEIBA                                                                              II  VII IX  X   Thrombin                                           Sample                                                                              (U/ml)                                                                             (U/ml)                                                                            (U/ml)                                                                            (U/ml)                                                                            (U/ml)                                                                            (U/ml)  A.sub.280                                  __________________________________________________________________________    Post-                                                                         activation                                                                          152.5                                                                              24.7                                                                              79.5                                                                              83.0                                                                              32.9                                                                              None detected                                                                         41.5                                       Pre-                                                                          activation                                                                          18.1 23.6                                                                              12.5                                                                              49.6                                                                              36.5                                                                              "       "                                          __________________________________________________________________________

The above eluate was filtered, sterile-filtered, and lyophilized (10 mlsamples). The dry product was reconstituted in water for injection (10ml for each sample) and analyzed according to the above assay methods.The results are outlined in Table 9B.

                  TABLE 9B                                                        ______________________________________                                                       Throm-   Solubility  Clar-                                     Sam-  FEIBA    bin      Time        ity.sup.a                                                                          Osmolarity                           ple   (U/ml)   (U/ml)   (Sec.) A.sub.280                                                                          (%)  (m0sm/kg)                            ______________________________________                                        Recon-                                                                              152      None     90     37.6 95   305                                  stituted       detected                                                       ______________________________________                                         .sup.a Clarity = Transmittance at 580 nanometers                         

EXAMPLE 10

The procedure of Example 7 was followed using two different calcium ionconcentrations, namely, 0.0005 M and 0.00075 M. The eluate was assayedat selected time intervals after addition of calcium ions thereto. Theresults are summarized in Table 10.

                  TABLE 10                                                        ______________________________________                                        Calcium ion concentration                                                     0.0005 M          0.00075 M                                                   Time FEIBA    Thrombin    FEIBA    Thrombin                                   (hrs)                                                                              (U/ml)   (U/ml)      (U/ml)   (U/ml)                                     ______________________________________                                        0     25      None detected                                                                              25      None detected                              17.5 173      0.6-1.0     174      0.7-1.1                                    19.5 186      "           206      "                                          22.0 243      0.8-1.5     255      1.2-1.8                                    24.0 270      "           298      "                                          ______________________________________                                    

EXAMPLE 11

The procedure of Example 7 was followed employing an A₂₈₀ =43 prior toaddition of calcium chloride. The eluate, after calcium ion addition,was analyzed at selected time intervals. Table 11 contains a summary ofthe results.

                  TABLE 11                                                        ______________________________________                                        Time         FEIBA    Thrombin                                                (hrs)        (U/ml)   (U/ml)                                                  ______________________________________                                        0            12       None detected                                           2.5          20.sup.b "                                                       5.0          23.sup.b "                                                       7.5          27.sup.b 0.48                                                    9.5          32.sup.b 0.56                                                    11.0         35.sup.b 0.78                                                    22           210      1.5                                                     24           241      1.9                                                     ______________________________________                                         .sup.a 1:20 dilution                                                          .sup.b It is to be noted that at least 11 hours are required before there     is a substantial increase in the amount of FEIBA substance in the eluate.

EXAMPLE 12

The procedure described in Example 7 was followed generally. Twodifferent calcium levels were employed, namely, 0.00025 M and 0.0005 M.In addition, the eluate was assayed after 19.75 hr., 22.75 hr., and25.75 hr. The results are outlined in Table 12.

                                      TABLE 12                                    __________________________________________________________________________    Calcium ion concentration                                                     0              0.00025     0.0005                                             Time                                                                             FEIBA                                                                              Thrombin                                                                             FEIBA                                                                              Thrombin                                                                             FEIBA                                                                              Thrombin                                      (hr.)                                                                            (U/ml)                                                                             (U/ml) (U/ml)                                                                             (U/ml) (U/ml)                                                                             (U/ml)                                        __________________________________________________________________________    19.75                                                                            29.5 <0.5   152  1.1    201  1.1                                           22.75                                                                            47.5 <0.5   225  1.1    260  4.2                                           25.75                                                                            53.0 <0.5   240  0.6    279  3.8                                           __________________________________________________________________________

The eluate after 25.75 hr. was filtered, sterile-filtered, andlyophilized. The dry product resolubilized in 40 sec. with a clarity of94% and A₂₈₀ =42.0.

EXAMPLE 13

The procedure of Example 7 was followed using calcium ion concentrationsof 0.00005 M, 0.00010 M, 0.00015 M, and no added calcium. The eluate wasassayed as described above with a summary of the results in Table 13.

                  TABLE 13                                                        ______________________________________                                        Calcium ion                                                                   Concentration   FEIBA                                                         (M)             (U/ml)                                                        ______________________________________                                        0                80                                                           0.00005         240                                                           0.00010         260                                                           0.00015         280                                                           ______________________________________                                    

EXAMPLE 14

The procedure of Example 7 was followed with the exceptions that (a) thecalcium ion concentration was 0.00025 M and (b) the pH was varied withinthe range 7.0-8.3. Three separate experiments (I-III) were conducted;the results are in Table 14.

                  TABLE 14                                                        ______________________________________                                        Experiment             FEIBA                                                  Number          pH     (U/ml).sup.a                                           ______________________________________                                         I              7.61   185                                                                    7.35   112                                                                    7.10   62                                                     II              7.60   233                                                                    7.70   210                                                                    7.81   210                                                                    7.89   242                                                    III             7.51   115                                                                    7.56   178                                                                    7.63   218                                                                    8.12   86                                                                     8.23   78                                                     ______________________________________                                         .sup.a The presence of thrombin could not be detected in any of the           samples.                                                                 

EXAMPLE 15

The method of Example 14 was followed with the exception that thetemperature and pH of the activation were varied. The results of twoseparate experiments are summarized in Table 15.

                  TABLE 15                                                        ______________________________________                                        Experiment           Temperature                                                                              FEIBA                                         Number    pH         (°C.)                                                                             (U/ml).sup.a                                  ______________________________________                                         II       7.61       10         185                                                     7.61       4.5        120                                                     7.61       23         140                                           II        7.60       10         233                                                     7.61       15.5       90                                                      7.71       15.5       90                                            ______________________________________                                         .sup.a The presence of thrombin could not be detected in any of the           samples.                                                                 

EXAMPLE 16

The procedure of Example 15 was employed with the exception that theeluate was diafiltered against 0.1 M sodium chloride alone. TRIS was notemployed during the diafiltration; however, the pH of the aqueous sodiumchloride was adjusted to 7.56, by addition of dilute hydrochloric acid.

For comparison, the procedure of Example 15 was followed to yield aFEIBA product.

The results are outlined in Table 16.

                  TABLE 16                                                        ______________________________________                                                       TRIS    FEIBA                                                  pH             (M)     (U/ml)                                                 ______________________________________                                        7.56           0       178                                                    7.63           0.05    218                                                    ______________________________________                                    

EXAMPLE 17

Effluent I (30 l.) was contacted with 300 g. of DEAE Sephadex gel andmixed together. After 2 hours the mixture was filtered to give 300 g. ofgel, which was washed sequentially with 3 l. of 0.2 M ammoniumbicarbonate, 2.1 l. of 0.3 M ammonium bicarbonate, and 3 l. of 0.2 Msodium chloride.

After washing, 2 l. of 0.55 M sodium chloride (I=0.55) was applied tothe gel to give an eluate (A₂₈₀ =12.80). The eluate was concentrated toA₂₈₀ of about 50 by ultrafiltration and diafiltered against 0.05 M TRISand 0.1 M sodium chloride (pH 7.6) buffer to a protein concentration ofA₂₈₀ =69.5.

The eluate was cooled to 10° C. for 24 hours. Then, Chelex®-100 resinwas mixed with the eluate to a level of 3% (w/v). The eluate wasfiltered to remove the resin, sterile-filtered, and lyophilized (10 ml.samples). The dry product was reconstituted in water for injection (10ml. for each lyophilized sample) and assayed according to theaforedescribed procedures.

For comparison the procedure of Example 7 was employed to prepare aproduct, which was filtered, sterile-filtered, lyophilized,reconstituted in water for injection as above, and assayed. The resultsare found in Table 17.

                  TABLE 17                                                        ______________________________________                                        Added                          Throm-                                         Acti- FEIBA    Factors (U/ml)  bin    NAPTT.sup.a                             vator (U/ml)   II    VII  IX  X   Xa   (U/ml) (sec.).sup.b                    ______________________________________                                        None   88      47    155  76  50  0.55 None   50                              0.0005                                 De-                                    M                                      tected                                 cal-  155      34    178  84  22  0.1  0.6    38                              cium                                                                          ion                                                                           ______________________________________                                         .sup.a NAPTT = Nonactivated Partial Thromboplastin time.                      .sup.b 1:100 dilution                                                    

EXAMPLE 18

Run A: The procedure of Example 7 was followed to prepare eluatecontaining FEIBA. The eluate (10 ml.) was lyophilized and thelyophilized material was reconstituted in 10 ml. of water for injection.The in vitro stability of the reconstituted product was observed at 0,2, and 4 hours.

Run B: The procedure of Example 17 was employed to yield a sample oflyophilized product, which was reconstituted in 10 ml. of water forinjection. The in vitro stability of the reconstituted material wasobserved as in Run A.

The results are summarized in Table 18.

                                      TABLE 18                                    __________________________________________________________________________    Activity (U/ml)                                                               0 hr.          2 hr.       4 hr.                                              Run                                                                              FEIBA                                                                              Thrombin                                                                             FEIBA                                                                              Thrombin                                                                             FEIBA                                                                              Thrombin                                      __________________________________________________________________________    A  290  0.7    290  0.5    290  0.5                                           B  83   N.D..sup.a                                                                           82   N.D..sup.a                                                                           89   N.D..sup.a                                    __________________________________________________________________________     .sup.a N.D. = None detected                                              

EXAMPLE 19

The procedure of Example 7 was followed except that the 0.2 M sodiumchloride wash was not discarded.

0.55 eluate was concentrated and diafiltered as in example 7, and the0.2 M sodium chloride wash was concentrated to an equal volume anddiafiltered against the same buffer as the 0.55 eluate.

Sample A: The 0.55 M eluate was diluted with an equal volume of 0.05 MTris chloride and 0.1 M NaCl.

Sample B: The 0.2 M wash was diluted similarly.

Sample C: Equal volumes of the 0.55 M eluate and the 0.2 M wash weremixed.

Calcium chloride was added to each sample (cooled to 10° C.) to aconcentration of 0.0005 M and each was held at 10° C. for 24 hours.Then, Chelex®-100 resin was mixed with each to a level of 10% (w/v). Thesuspensions were filtered to remove the resin.

The solutions were assayed as described above to give the results shownin Table 19.

                  TABLE 19                                                        ______________________________________                                                           FEIBA                                                      Sample             (U/ml)                                                     ______________________________________                                        A (0.55 M eluate)  76                                                         B (0.2 M wash)     <5                                                         C (A + B recombined)                                                                             14                                                         ______________________________________                                    

Having thus described the invention, we claim:
 1. A pharmaceuticalpreparation free of heparin, comprising coagulation Factors II, VII, IX,and X and having Factor VIII Inhibitor Bypassing Activity(FEIB-activity) wherein the FEIBA:Factor II ratio, the FEIBA:Factor VIIratio, the FEIBA:Factor IX ratio, and the FEIBA:Factor X ratio, areabout 10:1 to 0.1:1, respectively, and wherein the FEIBA:activity is atleast about 60 Units per milliliter and wherein the FEIBA:thrombin ratiois at least about 50:1.
 2. The preparation of claim 1 wherein theFEIBA:Factor Xa ratio is at least about 45:1.
 3. The preparation ofclaim 1 wherein the FEIBA:Factor II ratio is about 1.5:1 to 6:1, theFEIBA:Factor VII ratio is about 0.5:1 to 1.5:1, the FEIBA:Factor IXratio is about 1:1 to 2:1, and the FEIBA:Factor X ratio is about 1.5:1to 7:1.
 4. The preparation of claim 1 which contains less than 1.5 Unitsper millilter of thrombin activity.
 5. The preparation of claim 1 whichcontains less than 0.5 Units per milliliter of thrombin and theFEIBA:thrombin ratio is about 1000:1 or higher.
 6. A pharmaceuticalpreparation free of detectable thrombin and heparin, comprisingcoagulation Factors II, VII, IX, and X and having Factor VIII InhibitorBypassing Activity (FEIB-activity) wherein the FEIBA:Factor II ratio isabout 1.5:1 to 6:1, the FEIBA:Factor VII ratio is about 0.5:1 to 1.5:1,the FEIBA:Factor IX ratio is about 1:1 to 2:1, and the FEIBA:Factor Xratio is about 1.5:1 to 7:1.